SensoLyte® pNPP Protein Phosphatase Assay Kit *Colorimetric* -Note: activity assay kit to use later

Week 7 & 8

Mutant DNA Analysis

Week 5 & 6

DH5α Transformation with Mutant EhPTP pNIC-Bsa4

Figures 9-14. Transformation with 100 μl of product compared to a 1:10 dilution of the product in SOC growth media following site-directed mutagenesis with 25 ng of wild-type template DNA.

Figure 9. Transformation with EhMut125 produced 7,941 transformants, counted with ImageJ particle analysis (transformation efficiency = ___ cfu/μg)

Figure 10. Transformation with 1:10 EhMut125 produced 4,346 transformants, counted with ImageJ particle analysis (transformation efficiency = ___ cfu/μg)

Figure 11. Transformation with EhMut126 produced 11,151 transformants, counted with ImageJ particle analysis (transformation efficiency = ___ cfu/μg)

Figure 12. Transformation with 1:10 EhMut126 produced 5,114 transformants, counted with ImageJ particle analysis (transformation efficiency = ___ cfu/μg)

Figure 13. Transformation with EhMut125-126 produced 4,738 transformants, counted with ImageJ particle analysis (transformation efficiency = ___ cfu/μg)

Figure 14. Transformation with 1:10 EhMut125-126 produced 2,815 transformants, counted with ImageJ particle analysis (transformation efficiency = ___ cfu/μg)

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Nice work Kian. I assume the below sequences are just the unmodified plasmid? - DR. B 07052017
Yes, sample A and B of the wildtype EhPTP sequence (in pNIC28-Bsa4) from the start codon (green) to the pLIC-rev site (pink). I used DNA from sample A for mutagenesis on because it had an AvgQ score of 41, greater than that of sample B (34). Does the score difference necessarily matter? This afternoon, I plated transformed DH5-alpha with the control plasmid, EhMut125 (final and 1:10), EhMut126 (final and 1:10), and EhMut125-126 (final and 1:10) - each hopefully containing a Y to F codon substitution(s). Currently, the plates are incubating so tomorrow morning we'll be able to see if the mutants cultured!
- Kian 07/05/17

Week 3 & 4

DNA Analysis

Figure 8. Analyzed sequences of EhPTP DNA samples A and B from midiprep, sequenced by CORE with the pLIC-forward primer. The start codon (green), stop codon (red), polyhistidine-tag (yellow), and pLIC-reverse primer site (pink) are highlighted.

Midiprep

Figure 7. Nanodrop spectrophotometry of eluted DNA samples EhPTP-A and EhPTP-B. Sample A had an average concentration of 120.3 ng/μL and B had an average concentration of 138.4 ng/μL. Both samples had an average A260/280 of 2.15.

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Week 1 & 2

PyMOL Mutagenesis

PDB ID 3JS5

Figure 5.

Figure 6.

Figures 5 & 6. Mutagenesis of tyrosine residues 125 & 126 to non-phosphorylatable phenylalanine in PyMOL.
Note: Through Y to F substitution, the change of polarity due to the removal of the hydroxyl group (as seen above) should mimic the properties of Tyr residues 125 & 126 after undergoing dephosphorylation and their role in EhPTP activity, enabling a functional model of EhPTP pre- and post-phosphorylation of the two residues - individually and together - when compared to the activity of the wildtype strain in subsequent phosphatase assays.

Primer Design
Caplan_EhMut125-126, forward and reverse primers

Name_For/Rev	Oligo	Len	% GC	Tm	Ta*
Caplan_EhMut125-126_For	TCCGGACCCGttcttcGGTGGTGAAA	26	58	69	66
Caplan_EhMut125-126_Rev	ACTTCGGTGGTTTTGATGGTG	21	48	65	66

Required primers were designed using NEBaseChanger to substitute TACTAC (1054-1059), encoding Tyr 125 & 126, with TTCTTC to encode Phe 125 & 126.

Caplan_EhMut125, forward and reverse primers

Name_For/Rev	Oligo	Len	% GC	Tm	Ta*
Caplan_EhMut125_For	TCCGGACCCGttcTACGGTGGTG	23	65	72	68
Caplan_EhMut125_Rev	ACTTCGGTGGTTTTGATGGTGG	22	50	67	68

Required primers were designed using NEBaseChanger to substitute TAC (1054-1056), encoding Tyr 125, with TTC to encode Phe 125.

Caplan_EhMut126, forward and reverse primers

Name_For/Rev	Oligo	Len	% GC	Tm	Ta*
Caplan_EhMut126_For	GGACCCGTACttcGGTGGTGAAA	23	57	68	65
Caplan_EhMut126_Rev	GGAACTTCGGTGGTTTTGATG	21	48	63	65

Required primers were designed using NEBaseChanger to substitute TAC (1057-1059), encoding Tyr 126, with TTC to encode Phe 126.

PyMOL

PDB ID 3JS5

Figure 4.

Figure 3.

Residues of interest: Tyrosine 125 & 126, as discussed in the conclusion section of Linford and colleagues (2014).

• Reference: A.S. Linford et al. / Molecular & Biochemical Parasitology 193 (2014) 33–44
  • "The E. histolytica LMW-PTP protein sequence has the conserved DPYY loop containing the general acid/base asparagine and the two tyrosines that can be phosphorylated to regulate phosphatase activity or allow binding of adapter proteins"
  • "To address if phosphorylation of the DPYY loop tyrosines of the LMW- PTP affects its activity, for example, mutant forms of the LMW-PTP with substituted non-phosphorylatable residues at those positions could be expressed in vivo or in vitro and then assayed for phosphorylation and phosphatase activity; in vitro testing of these mutants alongside the recombinant wild-type LMW-PTP for phosphatase activity on pNPP after phosphorylation with Src kinase could be performed."

Amino acid substitution:

• Tyr to Phe 125 & 126
• 
• Phenylalanine is a known non-phosphorylatable, phosphotyrosine mimic

DH5α Transformation with EhPTP pNIC-Bsa4

Transformation with 0.03 μg of EhPTP produced 5,416 transformants, counted with ImageJ particle analysis (transformation efficiency = 181 x 10^3 cfu/μg).

Due to the lawn-like appearance of colonies, we surmised that the plate used for transformation with 0.003 μg of EhPTP did not contain kanamycin and thus transformants were not isolated.

Figure 2. Transformation with 0.0003 μg of EhPTP produced 49 transformants, counted with ImageJ particle analysis (transformation efficiency = 163 x 10^3 cfu/μg).

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Past EhPTP Work (Spring 2017)

BLAST Results

DNA Analysis

3JS5:A | PDBID | CHAIN | PROTEIN SEQUENCE
MAHHHHHHMGTLEAQTQGPGSMKLLFVCLGNICRSPAAEAVMKKVIQNHHLTEKYICDSAGTCSYHEGQQADSRMRKVGKSRGYQVDSISRPVVSSDFKNFDYIFAMDNDNYYELLDRCPEQYKQKIFKMVDFCTTIKTTEVPDPYYGGEKGFHRVIDILEDACENLIIKLEEGKLIN

PCR

Figure 9. In the third lane is a 1kb DNA ladder. The fourth, fifth, and sixth lanes show the products, or lack thereof, from amplification of varying plasmid concentrations of 3.0 ng/μl, 0.3 ng/μl, and 0.03ng/μl, respectively.

Mechanism schematic for PTP hydrolysis

Figure 1. Mechanism schematic for the hydrolysis reaction continuously catalyzed by EhPTP. Created with MarvinSketch molecular drawing software.